A new approach for studying intravasation of primary human breast cancer cells (PQ#24). Approximately 30-40% of women diagnosed with a curable breast cancer eventually die of metastatic disease. Since currently available technologies to study metastatic process are limited there is an urgent need to develop new approaches. We have been developing an approach for studying the intravasation step of metastasis composed of 3 integrated aspects: 1) devising an intravasation assay (INA), 2) optimizing methods for primary human breast cancer cells isolation from patient samples, and 3) adapting multiphoton imaging (MI) to explore cellular interactions leading to intravasation. Using these technologies we will explore a hypothesis that human breast cancers contain varying proportions of intravasation-competent cancer cells with distinct gene expression profile and they determine the clinical behavior of a particular breast cancer. Our preliminary studies indicate that: 1) we can use INA to study intravasation activity of breast cancer cells obtained by fine needle aspiration biopsy (FNA) from patients' tumors; 2) human breast cancers have varying proportions of intravasation-competent cells, and intravasate better in the presence of macrophages; 3) intravasation- competent primary human breast cancer cells express unique isoforms of an actin regulatory protein Mena that are related to an intravasation signature; 4) cancer cells with high MenaINV and low Mena11a isoform expression pattern participate with macrophages in transendothelial migration as assessed by INA and intravasation in mammary tumors in vivo, 5) intravasation sites called TMEM containing Mena overexpressing tumor cells and macrophages exist in human breast carcinomas and the number of TMEM sites correlates with distant metastasis. We also have evidence that some intravasation-competent cells adhere to apical endothelial surface upon intravasation while others do not. We propose to use INA and MI to study spatial and temporal interactions of human breast cancer cells with endothelial cells and macrophages as they intravasate in INA and assess estrogen, Her2Neu receptor and Mena isoform expression pattern in the intravasation- competent cells. In addition, we will correlate th clinicopathological parameters with the percentage of intravasation-competent cells that adhere to endothelial apical surface upon intravasation and those that do not, assess the effect of Mena isoform overexpression on intravasation and define a set of genes uniquely expressed in intravasation-competent human breast cancer cells. To assess if the ability of cancer cells to cross engineered endothelium in vitro reflects their in vivo intravasation potential we will collec fluorescent- labeled intravasation-competent and incompetent cells to determine differential interactions with endothelia, identify genes involved in endothelial interactions, and experimentally manipulate their intravasation activities in vitro and in vivo.